The hormone 1,25-dihydroxyvitamin D3 influences the growth anddifferentiation of a number of cell types. The functions of1,25-dihydroxyvitamin D3 are mediated through the vitamin D3receptor (VDR); therefore, an understanding of the regulationof VDR expression is important when considering the molecularmechanisms of differentiation induced by vitamin D3 and itsanalogues. ZEB, a Krüppel-type transcription factor knownto repress the transcription of several genes, binds to twosites within the VDR promoter and activates the transcriptionof this receptor in a cell-specific manner. Transfection ofZEB into SW620 colon carcinoma cells results in an up-regulationof the expression of endogenous VDR, confirming the role ofZEB in the transcriptional activation of the VDR gene. The expressionof VDR is also induced by c-MYB; thus, ZEB and c-MYB may modulatethe levels of VDR expression during differentiation in embryonaldevelopment, as well as in cancer cells.

VDR,3
a member of the steroid-thyroid receptor family, mediatesthe action of 1,25-OH2D3 by binding to vitamin D-responsiveelements and regulating gene transcription (1, 2)
. An understandingof the mechanisms regulating VDR gene expression is of importancebecause, e.g., nonfunctional VDR signaling has adverse effectson early postnatal development (3, 4, 5)
. Furthermore, becausethe levels of VDR expression correlate with the degree of differentiationand/or inhibition of growth of several malignant cell lines(6, 7, 8, 9)
, an understanding of the factors contributingto the expression of VDR is of particular importance to thepossible therapeutic use of vitamin D3 and its analogues asantiproliferative and/or differentiation agents.

The human protein ZEB is a transcription factor of the Krüppeltype with an internal homeodomain and zinc finger motifs atits amino and COOH termini (10)
. Homologues of ZEB includethe human variants AREB6(11)
and Nil-2a(12)
, the murineEF1/MEB1(13, 14)
, the rat Zfhep(15)
, the hamster BZP(16)
,the chicken EF1(17)
, and the Drosophila Zfh-1(18, 19)
.Henceforth, we refer to these proteins as ZEB homologues. Alignmentof the mouse, chicken, hamster, and human ZEB homologues revealsa high degree of conservation between all four species, with99.5% being within the zinc finger domains and 85% being outsideof these domains (14)
.

Histological analyses of chicken embryos established that themajor sites of ZEB expression are the notochord, myotome, limbbud, and neural crest derivatives (17)
. In mouse embryos, theexpression of ZEB is first detected in mesodermal tissues (i.e.,notochord, somite, and limb bud mesenchyme), as well as in neuralcrest derivatives (i.e., dorsal root ganglia and cephalic ganglia),and in parts of the central nervous system (i.e., hindbrainand motor neurons in the spinal cord; Ref. 20
).

ZEB and its homologues bind to subsets of E-boxes (most frequentlyto the sequence CACCTG), as well as to other sites which arenot E-boxes (10, 21)
. The E-boxes with the consensus sequenceCANNTG are major target sites for basic helix-loop-helix proteins,which induce the transcription of a variety of genes (22)
.However, by binding to E-boxes, ZEB and its homologues havebeen shown to repress the transcription of several genes, includingthe chicken -crystallin gene (17)
, the 4 integrin gene (23)
,the IL-2 gene (24)
, the GATA-3 gene (25)
, the CD4 gene (26)
,and others.

A role for ZEB in transcriptional activation has been suspected,based upon the presence of structural elements shown to be theactive domains of some transcriptional activators (27, 28, 29,30)
. E.g., ZEB has a long stretch of acidic amino acids (predominantlypoly-Glu) at the COOH terminus and a domain rich in prolinesin the middle (14, 17)
. To date, there are two reports thatsupport the possible role of ZEB as a transcriptional activator.Watanabe et al.(11)
demonstrated that AREB6, a human ZEB variant,activates transcription from the promoter of the rat Na,K-ATPase1subunit gene in a cell-specific manner; Chamberlain and Sanders(31)
demonstrated that EF1 up-regulates expression from thechicken ovalbumin gene.

In this report, we describe the presence of two E-boxes withinthe VDR promoter to which ZEB binds in vitro and demonstratethat exogenous expression of ZEB in COS 7 cells results in aconcentration-dependent up-regulation of VDR promoter activity.Optimal up-regulation of the VDR promoter by ZEB required itsinteraction with both binding sites. The transcriptional activationof the VDR promoter by ZEB was not influenced by the presenceof cofactors such as CtBP and CBP, although direct binding betweenZEB and CtBP has been reported (32, 33, 34)
. We have also establishedthat the VDR promoter is c-MYB inducible. Unlike other c-MYB-induciblepromoters which can be repressed by ZEB (26, 35)
, the coexpressionof ZEB and c-MYB in COS 7 cells induced the VDR reporter genein an additive fashion.

Binding of ZEB to Sequences of the VDR Promoter.
Analysis of the murine VDR promoter sequence (36)
revealedthe presence of two E-boxes, both with the consensus sequenceCACCTG and shown to be the target for ZEB binding (10)
. Thehuman VDR promoter sequence, which shows little sequence homologyto the murine promoter region, also has two CACCTG E-boxes (37)
.The presence of potential ZEB binding sites in both speciesimplies that an evolutionary conserved mechanism exists forregulation of the expression of the VDR gene. Therefore, asan initial experiment, we tested these two sites for their abilityto bind to ZEB in vitro. Fig. 1
, Lanes 2 and 6 shows that bothmurine probes containing the two CACCTG boxes were capable ofbinding the COOH-terminal zinc fingers of ZEB. Competition withunlabeled probe abrogated the formation of a detectable DNA-proteincomplex (Fig. 1
, Lanes 3 and 7), thereby confirming the specificityof the binding. In addition, probes in which the E-boxes weremutated did not bind to recombinant ZEB protein (Fig. 1
, Lanes4 and 8), indicating that the CACCTG sequence is the targetsite for ZEB.

Fig. 1. Binding of ZEB to the two E-boxes present in the VDR promoter region. GMSAs were performed as described in "Materials and Methods" using digoxigenin-labeled probes containing the Z1 site (Lanes 14) or the Z2 site (Lanes 58). Lanes 1 and 5, the probes alone; Lanes 2 and 6, the probes incubated with recombinant ZEB protein; Lanes 3 and 7, the same binding reactions as in Lanes 2 and 6, except that a 100-fold molar excess of unlabeled probe was added; and Lanes 4 and 8, the binding reactions of probes with mutated E-boxes and recombinant ZEB protein. Arrow, the ZEB-binding complex. A representative gel of the GMSA is shown.

Fig. 2. Up-regulation of transcription from the murine VDR promoter induced by ZEB. In A, transcriptional activity of the VDR promoter fused to the luciferase gene in the pGL3Basic vector was assayed by cotransfection of this reporter gene (0.2 µg/well) with increasing amounts of ZEB expression vector (0.125, 0.25, and 0.5 µg) in COS 7 cells. B, requirement of CACCTG boxes in the VDR promoter region for the activation of the VDR promoter by ZEB in COS 7 cells. The activity of the wild-type VDR promoter reporter gene (0.2 µg/well) was measured in the presence of 0.5 µg of ZEB expression vector per well (ZEB) or the presence of 0.5 µg of control vector per well (CTRL) and compared with the activities of mutated VDR reporter genes with only one mutated E-box (Z1 or Z2) or with mutations in both E-boxes (Z1,2) in the presence of 0.5 µg of ZEB expression vector per well. Luciferase activities were normalized against the activities of the control vector pRL-TK. The average of three to seven independent transfections each with triplicate samples and SDs are shown. In some cases, the SDs were very low and do not show up as observable error bars.

To ascertain whether the two CACCTG E-boxes present in the VDRpromoter were required to mediate the ability of ZEB to inducethe expression of the VDR reporter gene, we mutated each ofthese sites. Mutated VDR promoter constructs, with a changein the first E-box at 10341039 nt (Z1), in the secondE-box at 11291134 nt (Z2), or at both sites (Z1,2), werecotransfected with a fixed amount of ZEB expression plasmidin COS 7 cells and assayed for luciferase activity. MutationZ1 resulted in an 50% decrease in VDR reporter activity, whereasmutation Z2 produced an 40% decrease (Fig. 2B)
. These resultssuggest that both E-boxes are involved in the induction producedby ZEB. This conclusion was further supported by the fact thatwhen the double mutant Z1,Z2 was cotransfected with the ZEBexpression vector, its activity was similar to that of the wild-typeVDR reporter gene in the absence of ZEB expression (Fig. 2B)
.

The induction of the rat Na,K-ATPase1 subunit gene by AREB6,a human ZEB variant, has been reported to be cell specific (11)
.To determine whether the induction of the VDR promoter by ZEBis cell specific, we performed VDR reporter gene/ZEB cotransfectionexperiments in human colon and prostate carcinoma cells in which1,25-OH2D3 has been shown to inhibit proliferation (38, 39,40)
. In the SW620 colon carcinoma cell line, exogenous ZEBexpression induced VDR reporter gene activity by 2-fold; however,because the control pGL3Basic vector decreased activity in thepresence of ZEB, the relative increase in VDR reporter geneactivity was approximately 4.7 ± 0.4-fold (Fig. 3A)
.In LNCaP prostate cancer cells, the expression of exogenousZEB had no significant effect on both the pGL3Basic and VDRreporter gene, whereas in HCT-116 colon carcinoma cells, thecotransfection with ZEB resulted in an 2-fold increase in bothpGL3Basic and VDR reporter gene transcriptional activities;therefore, the ratio of these two activities was close to 1.0(Fig. 3A)
. Among the cell lines used, SW620 cells exhibitedthe highest endogenous steady-state levels of ZEB and VDR mRNAexpression (Fig. 3B)
; therefore, it is most probable that celltype characteristics, rather than ZEB expression levels, contributeto the observed differences in ZEB transcriptional activityon the VDR reporter gene.

Fig. 3. Cell-specific activation of the VDR promoter by ZEB. In A, HCT-116, SW620, and LNCaP cells were transiently transfected with 0.2 µg/well of VDR reporter gene or control vector pGL3Basic in the absence or presence of 0.5 µg of ZEB expression vector. The results represent the change in transcriptional activity in the VDR reporter gene in the presence of ZEB normalized to that of the pGL3Basic control vector. The average of three independent transfections and SDs are shown. In B, Northern blot analyses for the expression of ZEB, VDR, and actin were performed as described in "Materials and Methods" for LNCaP (Lane 1), HCT-116 (Lane 2), and SW620 (Lane 3) cells.

We wished to determine whether the endogenous VDR gene is up-regulatedby ZEB overexpression in SW620 colon carcinoma cells. Attemptsto stably transfect SW620 cells with a ZEB expression vectorwere unsuccessful, and no ZEB-expressing colonies were obtainedfrom G418-resistant cells (data not shown), possibly becauseZEB overexpression selects against cell proliferation. Therefore,SW620 cells were transiently transfected with a ZEB expressionvector. This transfection resulted in an 50% increase in endogenousVDR protein steady-state levels as estimated by Western blotanalyses (Fig. 4)
. The observed up-regulation of VDR proteinby ZEB in transiently transfected SW620 cells was derived froma cell pool in which only a fraction of the total cell numberwas successfully transfected. To establish the fraction of SW620cells transfected, we used a ß-galactosidase expressionvector (CMV-Gal) to evaluate transient transfection efficiency("Materials and Methods") and observed that 11.6 ± 0.8%of the SW620 cell population was transfected with CMV-Gal. Assuminga similar transfection efficiency with the ZEB expression vector,these findings suggest that transfection of 12% of SW620 cellsby the ZEB expression vector resulted in the observed 50% up-regulationof endogenous VDR protein steady-state levels (Fig. 4)
andthat the up-regulation of VDR protein levels in those cellstransfected with ZEB is higher than the 50% increase determinedfrom the analysis of total cell lysates.

Fig. 4. Endogenous VDR expression in SW620 cells transfected with ZEB expression vector. SW620 cells were transiently transfected with 6 µg of control vector (Lanes 1 and 2) or 6 µg of ZEB expression vector (Lanes 3 and 4). Cells were harvested at 48 h after transfection. Total protein extraction and Western blot analyses were performed as indicated in "Materials and Methods." The experiment was repeated twice with duplicate samples, and a representative Western blot is shown.

Induction of VDR Promoter Activity by c-MYB.
ZEB has been reported to negatively regulate both myogenesisand hematopoiesis by repressing genes that control these differentiationprocesses (35)
. These authors proposed that activation of hematopoieticgenes in the presence of ZEB is achieved by an unidentifiedmechanism requiring the presence of both the c-MYB and Ets transcriptionfactors. The activation of the CD4 promoter in the presenceof ZEB has also been reported to require both c-MYB and Ets(26)
. Analysis of the sequence of the mouse VDR promoter revealedthat there are five potential c-MYB binding sites. Cotransfectionof COS 7 cells with VDR reporter gene construct and increasingamounts of c-MYB expression construct resulted in a linear (15-fold)increase in VDR promoter activity (Fig. 5A)
. This finding suggeststhat c-MYB is another transcription factor with the potentialto regulate VDR promoter activity. In addition, we confirmedthat CBP is a coactivator with c-MYB (41)
in the context ofthe VDR promoter (Fig. 5B)
. The up-regulation of endogenousVDR expression by c-MYB was confirmed by transfection experimentsin WEHI-3B D+ murine myelomonocytic leukemia cells, U-937 humanhistiocytic lymphoma cells, and HL-60 human promyelocytic leukemiacells. In all of these cell lines, exogenous c-MYB expressionresulted in an 50% increase in endogenous VDR protein steady-statelevels (Fig. 6)
, thereby providing further evidence that theVDR gene is regulated by c-MYB.

Fig. 5. Transcription from the VDR reporter gene induced by c-MYB. In A, COS 7 cells were transiently transfected with 0.2 µg of VDR reporter gene construct and with increasing amounts of c-MYB expression construct (0.3, 0.5, and 0.8 µg). Luciferase activities were normalized against the activities of the control vector pRL-null. B, coactivation of the induction of the VDR promoter by c-MYB and CBP. COS 7 cells were transiently transfected with 0.2 µg of VDR reporter gene construct and c-MYB (0.3 µg), CBP (0.3 µg), or the combination of c-MYB and CBP expression vectors. Luciferase activity was assayed at 48 h after transfection. Luciferase activities of the VDR reporter construct were normalized against the activities of the pGL3Basic vector alone, because CBP has an inducing effect on the control itself. C, additive induction of the transcription from the VDR promoter by c-MYB and ZEB. COS 7 cells were transiently transfected with 0.2 µg of VDR reporter gene construct and c-MYB (0.3 µg), ZEB (0.3 µg), or the combination of c-MYB and ZEB expression vectors. Luciferase activity was assayed at 48 h after transfection. Luciferase activities were normalized against the activities of the control vector pRL-null. The average of three independent transfections and SDs are shown. Experiments with very small SDs do not show observable error bars.

Fig. 6. Up-regulation of the endogenous expression of VDR by c-MYB. WEHI-3B D+ (A), U-937 (B), and HL-60 (C) cells were transiently transfected with 4 µg of control vector (CTRL) or with 4 µg of c-MYB expression vector (MYB). Cells were harvested at 48 h, and Western blot analyses were performed as described in "Materials and Methods." The transfections were repeated twice with duplicate samples; representative Western blots are shown.

Because the VDR promoter is up-regulated by ZEB, we reasonedthat a possible role for ZEB in repressing VDR promoter activitymight be detected only if ZEB competes with another transcriptionalactivator of VDR promoter activity, such as c-MYB. However,cotransfection experiments with c-MYB and ZEB in COS 7 cellsresulted in a supra-additive up-regulation of VDR promoter expression(Fig. 5C)
.

The VDR Is a Ligand-dependent Transcription Factor that Mediates the Regulation of Gene Expression by 1,25-OH2D3.
A major function of 1,25-OH2D3 is the maintenance of physiologicallevels of calcium and phosphate in the plasma (42)
. However,new functions for 1,25-OH2D3 have been considered after theVDR was localized in a variety of cell types and after 1,25-OH2D3was identified as a factor which influences cellular proliferationand differentiation (7, 43, 44)
. The regulation of the expressionof the VDR gene is of particular interest because a correlationhas been established between steady-state levels of VDR andthe ability of 1,25-OH2D3 to influence cell growth and differentiation(6, 7, 8, 9)
. In addition, by enhancing the stability of theVDR protein, 1,25-OH2D3 can initiate a positive feedback loopwhich may enhance differentiation (45, 46)
. The ability ofthe Sp1 and WT1 transcription factors to up-regulate the expressionof the VDR gene has been demonstrated (47, 48)
.

In the present study, we report that another transcriptionalfactor, ZEB, up-regulates the activity of the VDR promoter bybinding to two E-boxes within this promoter. Although it ispossible that ZEB enhances VDR promoter activity indirectly,through enhanced expression of transcriptional activators ofthe VDR gene or by blocking the binding of a transcriptionalrepressor(s) to the VDR promoter, our data are most consistentwith a direct effect of ZEB on VDR promoter activity. The conceptof direct activation of the VDR gene by ZEB is supported bythe ability of ZEB to bind specifically to two E-box-containingVDR promoter sequences in vitro (Fig. 1)
. However, is it possiblethat ZEB blocks repression of the VDR promoter, instead of directlyactivating it? A VDR promoter construct encompassing the first500 bp upstream of the transcriptional start site exhibits expressionlevels higher than that of the construct with an additional1000 bp upstream (36)
. One possible interpretation of thisfinding is that a transcriptional repressor targets sequencesclose to the two ZEB (Z1 and Z2) sites we have examined. Ifsuch a repressor exists, ZEB may block its binding and, therefore,its repressive function. Direct competition between ZEB anda putative repressor for binding to the same E-boxes is unlikely;otherwise, we would have observed similar levels of expressionfrom the short (0.5 kb) VDR construct (which lacks both theZ1 and Z2 E-boxes) and from the mutant Z1,2 VDR construct (1.5kb). Rather, the mutant Z1,2 VDR construct (1.5 kb) is expressedat levels similar to that of the wild-type 1.5-kb VDR constructin the absence of ZEB coexpression (Fig. 2B)
.

It has been reported that ZEB represses the transcription ofsome promoters through interaction with the corepressor CtBP(33, 34)
. Our findings establish that chicken ZEB can alsoactivate gene expression, and because the role of ZEB in transcriptionalactivation is cell specific, we reasoned that the dual roleof ZEB in repression and activation may depend not only uponthe promoter sequences but also upon the association of ZEBwith different cofactors. The corepressor CtBP has been shownto interact with a wide variety of transcription factors withdual roles, such as BKLF (32, 49)
, AREB6 (11)
, and Evi-1(50)
. However, CtBP1 and CtBP2 cotransfection experiments withZEB in COS 7 cells did not produce a change in the up-regulationof VDR promoter activity by ZEB (data not shown). Therefore,the activating function of ZEB on the VDR promoter is not attributableto a lack of or low steady-state level of CtBP in COS 7 cells.We also have examined whether the activation of the VDR promoterby ZEB is dependent upon the presence of the transcriptionalcoactivator CBP. Cotransfection of ZEB and CBP resulted in anadditive up-regulation of the VDR reporter gene; however, transfectionwith CBP alone increased transcription from the VDR reportergene, as well as from the control pGL3Basic vector (data notshown). Therefore, the specificity of the CBP/ZEB coactivationwas not confirmed.

Unlike c-MYB inducible promoters whose expression is down-regulatedby ZEB (26)
, we have found that expression from the VDR promoteris up-regulated in an additive fashion by c-MYB and ZEB in COS7 cells (Fig. 5C)
. c-MYB transfections also induced endogenousVDR gene expression (Fig. 6)
in several cell types, includingWEHI-3B D+ myelomonocytic leukemia cells that can differentiatealong the granulocytic pathway (51)
or the monocytic pathway(52)
, U-937 histiocytic lymphoma cells that can be inducedto differentiate into monocyte-like cells (53)
, and HL-60 promyelocyticleukemia cells that can differentiate into monocyte- (54)
orneutrophil-like cells (55, 56)
. The increase in the steady-statelevels of the VDR protein in c-MYB transfected cells and theability of c-MYB to induce the VDR reporter gene in transienttransfections argue that c-MYB is a transcriptional regulatorof the VDR gene. The precise mechanism (direct or indirect)by which c-MYB influences VDR gene expression is the subjectof future experimental work.

The activation of VDR gene expression by c-MYB is not surprisingbecause, in addition to the role of c-MYB in the proliferationof immature hematopoietic cells (57, 58)
, c-MYB has been alsoimplicated in several differentiation pathways (59, 60)
. Thecontrast between the reported opposing effects of ZEB and c-MYBon transcriptional activity (26)
and our observation of cooperativitybetween ZEB and c-MYB (Fig. 5C)
is most likely related to therole of ZEB as a transcriptional activator, rather than as arepressor, in our system. Therefore, instead of the requirementfor c-MYB and/or Ets to overcome ZEB-induced transcriptionalrepression (26, 35)
in our system, both ZEB and c-MYB positivelystimulate VDR promoter activity and supra-additively enhanceexpression. These different interactions of ZEB and c-MYB maybe promoter and cell-type specific and may be important in modulatingthe expression of genes involved in decisions of proliferationversus differentiation.

The expression of ZEB in embryogenesis suggests that this transcriptionfactor regulates VDR expression levels starting in early developmentwhen initial differentiation takes place, a concept which isconsistent with a role for 1,25-OH2D3 in differentiation. Thebeginning of ZEB expression coincides with the beginning oforganogenesis (17)
. ZEB is primarily expressed in the mesoderm(17, 20)
, a layer of the postgastrulation embryo that givesrise to cartilage, bone, fibrous tissue, muscle cells, and partsof the urogenital system, as well as the vascular system, includingblood cells. Coincidentally, 1,25-OH2D3 has been demonstratedto play a role in the formation of bone and cartilage (61, 62,63)
, in the induction of immature myeloid cells toward monocyte/macrophages,and in the differentiation of small intestine (64)
. Therefore,the expression of ZEB may well be necessary for the functionof 1,25-OH2D3 in the differentiation of several cell types.

We hypothesize that ZEB is a transcriptional regulator of theVDR gene in vivo, a concept supported by similarities in thephenotypes of ZEB and VDR knockout mice. Both ZEB null mutantmice (20)
and VDR null mutant mice (65)
exhibit signs of growthretardation and abnormalities in skeletal elements. On the basisof the findings described in the present report, we speculatethat the lack of ZEB expression in ZEB knockout mice resultsin relatively low steady-state levels of the VDR at particulardevelopmental stages and thus contributes to the skeletal deformitiesobserved in these animals. The fact that ZEB null mutants exhibitmuch more severe bone abnormalities and that the onset of growthretardation is earlier than that in VDR knockout mice furthersuggests that ZEB regulates the expression of not only the VDRbut also of other genes which contribute to skeletal developmentin embryogenesis. E.g., we have found that the activity of themurine osteocalcin 2 promoter is also modulated by ZEB.4

In summary, taking into account our findings, as well as thosein previous reports, that ZEB functions as either a transcriptionalrepressor (17, 23, 24, 25, 26)
or as a transcriptional activator(11, 31)
in the context of different promoters, we concludethat the role of ZEB as a transcriptional factor is promoterand cell-type dependent. Requirements for particular structuralelements of the promoter region, the presence of different proteinpartners for ZEB, the presence of transcriptional partners competingwith ZEB for binding sites, and/or the possibility that differentposttranscriptional modifications of ZEB exist are all factorswhich may determine whether ZEB acts in a repressive or activatingmode for gene transcription.

Transfection, Luciferase Assay, and Determination of Transfection Efficiency.
Transfections were performed with cells plated in 24-well dishesat 72 h before transfection (unless specified otherwise) usingthe GenePorter transfection reagent, according to the protocolof the manufacturer (Gene Therapy Systems, Inc., San Diego,CA). Briefly, reporter gene constructs were cotransfected withindicated amounts of various expression vectors. After dilutingDNA and transfection reagent with serum-free medium, the twosolutions were combined, and complexes were allowed to formfor 45 min at room temperature. Cells were incubated with DNA-GenePortermixture for 5 h before the addition of medium containing doublethe regular concentration of FBS (as described in the "Materialsand Methods Cell Culture" section). Luciferase reporter assayswere performed at 24 or 48 h after transfection, following theprotocol of Promega Corp. To normalize for transfection efficiency,pRL-TK or pRL-null vectors (Promega Corp.) were cotransfected.However, because the expression of certain proteins influencesthe expression of pRL-TK, and even that of pRL-null in sometransfections, the protein concentration, determined by themethod of Bradford (67)
, was used to normalize the values forluciferase activity. To determine the transfection efficiencyof the GenePorter-SW620 cell system, SW620 cells were transfectedas described above with either 1 µg of CMV-Gal or pCINeo(Promega Corp.); three wells of cells were transfected witheach plasmid. Cells were then processed for ß-galactosidasestaining with the PanVera (Madison, WI) ß-galactosidaseStaining Kit, according to manufacturers protocols. Atotal of 300 cells/well were counted, and the percentage ofblue cells was determined.

Northern Blot Analyses.
Total RNA was isolated with Trizol reagent (Life Technologies,Inc., Gaithersburg, MD), according to the protocol of the manufacturer,and poly(A) RNA was selected with Oligotex mRNA kit (Qiagen,Valencia, CA). For Northern blot analyses, 1.5 µg of poly(A)RNA were separated on 1% formaldehyde denaturing gel and transferredto nylon membranes. Probes for ZEB, VDR, and actin were preparedfrom the cDNAs for chicken ZEB, human VDR, and chicken actin,respectively. Probes were labeled with the Random Primers DNAlabeling system of Life Technologies, Inc. using [-32P]-dCTP.Prehybridizations and hybridizations were carried out usingRapid-hyb buffer (Amersham Pharmacia Biotech, Piscataway, NJ).

The costs of publication of this article were defrayed in partby the payment of page charges. This article must thereforebe hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

1 Supported in part by U. S. Public Health Service Grant CA-02817from the National Cancer Institute.

2 To whom requests for reprints should be addressed, at Departmentof Pharmacology, Yale University School of Medicine, 333 CedarStreet, New Haven, CT 06520. Phone: (203) 785-4533; Fax: (203)737-2045; E-mail: alan.sartorelli{at}yale.edu